MerR Family Research Articles

Crystal structure of the [2Fe-2S] oxidative-stress sensor SoxR bound to DNA

The [2Fe-2S] transcription factor SoxR, a member of the MerR family, functions as a bacterial sensor of oxidative stress such as superoxide and nitric oxide. SoxR is activated by reversible one-electron oxidation of the [2Fe-2S] cluster and then enhances the production of various antioxidant proteins through the soxRS regulon. In the active state, SoxR and other MerR family proteins activate transcription from unique promoters, which have a long 19- or 20-bp spacer between the -35 and -10 operator elements, by untwisting the promoter DNA. Here, we show the crystal structures of SoxR and its complex with the target promoter in the oxidized (active) state. The structures reveal that the [2Fe-2S] cluster of SoxR is completely solvent-exposed and surrounded by an asymmetric environment stabilized by interaction with the other subunit. The asymmetrically charged environment of the [2Fe-2S] cluster probably causes redox-dependent conformational changes of SoxR and the target promoter. Compared with the promoter structures with the 19-bp spacer previously studied, the DNA structure is more sharply bent, by approximately 1 bp, with the two central base pairs holding Watson-Crick base pairs. Comparison of the target promoter sequences of the MerR family indicates that the present DNA structure represents the activated conformation of the target promoter with a 20-bp spacer in the MerR family.

Glutathione-Dependent Alcohol Dehydrogenase AdhC Is Required for Defense against Nitrosative Stress in Haemophilus influenzae

In Haemophilus influenzae Rd KW20, we identified a gene, adhC, which encodes a class III alcohol dehydrogenase (AdhC) and has S-nitrosoglutathione reductase activity. adhC exists on an operon with estD, which encodes an esterase. Divergent to the adhC-estD operon is the Haemophilus influenzae nmlR gene (nmlR(HI)), which encodes a MerR family regulator that is homologous to the Neisseria MerR-like regulator (NmlR). Analysis of an nmlR(HI) mutant indicated that expression of the adhC-estD operon is regulated by NmlR(HI) in strain Rd KW20. Chromosomal inactivation of either adhC or nmlR(HI) resulted in sensitivity to S-nitrosoglutathione and decreased S-nitrosoglutathione reductase activity. Examination of the NmlR(HI)-AdhC system in the genome sequences of nontypeable H. influenzae strains R2846, R2866, and 86-028NP identified significant variations. The adhC gene of 86-028NP was predicted to be nonfunctional due to a premature stop codon. Polymorphisms in the operator/promoter region of R2866 resulted in reduced enzyme activity. This correlated with an increased sensitivity to S-nitrosoglutathione. The adhC-nmlR(HI) system was examined in thirty-three clinical isolates (both capsular and nontypeable strains). Nucleic acid sequence data showed that only strain 86-028NP contained a premature stop codon. There were some variations in the DNA sequence of the operator/promoter region which altered the nmlR(HI) promoter. However, the clinical isolates still possessed S-nitrosoglutathione reductase activity and showed at least the equivalent ability to grow in the presence of S-nitrosoglutathione as Rd KW20. These data suggest that the nmlR(HI)-adhC system has a role in the defense against nitrosative stress in Haemophilus influenzae.

19F-NMR Reveals Metal and Operator-induced Allostery in MerR

Metalloregulators of the MerR family activate transcription upon metal binding by underwinding the operator-promoter DNA to permit open complex formation by pre-bound RNA polymerase. Historically, MerR's allostery has been monitored only indirectly via nuclease sensitivity or by fluorescent nucleotide probes and was very specific for Hg(II), although purified MerR binds several thiophilic metals. To observe directly MerR's ligand-induced behavior we made 2-fluorotyrosine-substituted MerR and found similar, minor changes in 19F chemical shifts of tyrosine residues in the free protein exposed to Hg(II), Cd(II) or Zn(II). However, DNA binding elicits large chemical shift changes in MerR's tyrosine residues and in DNA-bound MerR Hg(II) provokes changes very distinct from those of Cd(II) or Zn(II). These chemical shift changes and other biophysical and phenotypic properties of wild-type MerR and relevant mutants reveal elements of an allosteric network that enables the coordination state of the metal binding site to direct metal-specific movements in the distant DNA binding site and the DNA-bound state also to affect the metal binding domain.

Design of an Emission Ratiometric Biosensor from MerR Family Proteins: A Sensitive and Selective Sensor for Hg2+

A ratiometric fluorescent biosensor for Hg2+ is constructed from the MerR protein and a duplex DNA containing a pyrene excimer.

A Galvanizing Story—Protein Stability and Zinc Homeostasis

Zinc is an essential component of protein function in all living cells. The intracellular availability of zinc ions must be balanced against their potential toxicity. Maintaining optimal zinc ion levels requires the integration of zinc enzyme expression with zinc transport. Much of the effort to understand bacterial zinc homeostasis, as well as that of other metals, has focused on transcriptional regulation and the biochemical properties of the relevant transcriptional regulators. Less is known about the translational and posttranslational mechanisms of metal homeostasis in bacteria, although these are clearly important (9–11). The significance of protein stability and turnover is emphasized in the results of a study of the zinc-dependent ZntR transcriptional activator, which are presented in this issue (15). These new data provide a way to reconcile the results of previous biochemical and biological studies of ZntR function (6, 13, 18) and provide a better model for understanding zinc homeostasis and one which can be applied to other metals. This study underscores the importance of designing experiments that account for the dynamic nature of cell physiology and its effect on intracellular transition metal utilization. There are many known zinc proteins in Escherichia coli (50), several of which are essential (e.g., RNA polymerase and tRNA synthetases). Growth conditions that change the level of zinc protein expression will require adjustments of intracellular zinc levels to ensure that sufficient metal is available for the generation of active proteins. Access to zinc will depend on the concentration of both extracellular zinc and that recovered from the turnover of other Zncontaining proteins. Zinc ions are transported into the cytoplasm via ZnuABC (14), although other import pathways exist (5). ZnuABC expression is regulated by Zur (14), a Zn-dependent transcriptional repressor homologous to the well-known Fur family of metal-dependent regulators (8). When Zur binds available intracellular zinc, it represses ZnuABC transcription, ultimately leading to cessation of zinc import via this pathway. Excess zinc ions are exported by the zinc-inducible exporter ZntA (1, 16), whose expression is regulated by ZntR (2), a member of the MerR family of transcriptional regulators (7).

MerR family transcription activators: similar designs, different specificities

Living organisms use metals for a variety of essential functions, and face the problems of how to acquire and regulate the intracellular levels of those metals they need, differentiate between essential and toxic metals, and remove from the cell or detoxify metals that are toxic. In bacteria, cytoplasmic metal ion responsive transcriptional regulators are important in regulating the expression of genes involved in metal ion homeostasis and efflux systems. The MerR family of transcriptional activators are metal sensing regulators that are found in different bacteria and have a common design, but have evolved to recognize and respond to different metals. In this issue of Molecular Microbiology, work by Checa and colleagues describes for the first time a gold-specific MerR family regulator named GolS from Salmonella enterica serovar Typhimurium that controls the production of an efflux pump and a metal chaperone protein that confer resistance to Au salts.

Bacterial sensing of and resistance to gold salts

The MerR family is a group of bacterial transcriptional regulators that respond to different environmental stimuli, such as heavy metals, oxidative stress or antibiotics. Here we characterize a new member of this family that is highly selective for Au ions. We show that this Salmonella regulator, named GolS, directly controls the expression of at least two transcriptional units specifically required for Au resistance. By chromosomal mutagenesis, we demonstrated that Au-selectivity is accomplished by a metal-binding motif in GolS. Among the monovalent metal-ion sensing MerR regulators GolS clusters in a branch distant from enterobacterial CueR orthologues. We propose that GolS and its homologues evolved to cope with toxic concentration of Au ion, allowing microorganisms to withstand contaminated environments.

Crystallization and preliminary X-ray crystallographic studies of the oxidative-stress sensor SoxR and its complex with DNA

SoxR, a member of the MerR family of transcriptional activators, functions as a sensor of oxidative stress. The redox states of the 2Fe-2S cluster of SoxR regulate the activity of SoxR. Here, the crystallization and preliminary crystallographic analysis of SoxR and its complex with DNA are reported. Crystals of SoxR were obtained using PEG 10,000 and glycerol as precipitants. The crystals of SoxR belong to space group P6(2) or P6(4), with unit-cell parameters a = b = 80.0, c = 88.1 A. Crystals of the SoxR-DNA complex were obtained using a 20 bp DNA fragment from a condition containing PEG 10,000 and sodium/potassium tartrate. The crystals of the SoxR-DNA complex belong to space group P6(1)22 or P6(5)22, with unit-cell parameters a = b = 53.5, c = 355.6 A. Diffraction data were collected to a maximum resolution of 3.2 and 2.7 A for SoxR and the SoxR-DNA complex, respectively.

Functional Analysis of the Carboxy-Terminal Region of Bacillus subtilis TnrA, a MerR Family Protein

The Bacillus subtilis TnrA transcription factor belongs to the MerR family of proteins and regulates gene expression during nitrogen-limited growth. When B. subtilis cells are grown with excess nitrogen, feedback-inhibited glutamine synthetase forms a protein-protein complex with TnrA that prevents TnrA from binding to DNA. The C-terminal region of TnrA is required for the interaction with glutamine synthetase. Alanine scanning mutagenesis of the C-terminal region of TnrA identified three classes of mutants that altered the regulation by glutamine synthetase. While expression of the TnrA-regulated amtB gene was expressed constitutively in the class I (M96A, Q100A, and A103G) and class II (L97A, L101A, and F105A) mutants, the class II mutants were unable to grow on minimal medium unless a complex mixture of amino acids was present. The class III tnrA mutants (R93A, G99A, N102A, H104A, and Y107A mutants) were partially defective in the regulation of TnrA activity. In vitro experiments showed that feedback-inhibited glutamine synthetase had a significantly reduced ability to inhibit the DNA-binding activity of several class I and class II mutant TnrA proteins. A coiled-coil homology model of the C-terminal region of TnrA is used to explain the properties of the class I and II mutant proteins. The C-terminal region of TnrA corresponds to a dimerization domain in other MerR family proteins. Surprisingly, gel filtration and cross-linking analysis showed that a truncated TnrA protein which contained only the N-terminal DNA binding domain was dimeric. The implications of these results for the structure of TnrA are discussed.

Comparative genomics of regulation of heavy metal resistance in Eubacteria

BackgroundHeavy metal resistance (HMR) in Eubacteria is regulated by a variety of systems including transcription factors from the MerR family (COG0789). The HMR systems are characterized by the complex signal structure (strong palindrome within a 19 or 20 bp promoter spacer), and usually consist of transporter and regulator genes. Some HMR regulons also include detoxification systems. The number of sequenced bacterial genomes is constantly increasing and even though HMR resistance regulons of the COG0789 type usually consist of few genes per genome, the computational analysis may contribute to the understanding of the cellular systems of metal detoxification.ResultsWe studied the mercury (MerR), copper (CueR and HmrR), cadmium (CadR), lead (PbrR), and zinc (ZntR) resistance systems and demonstrated that combining protein sequence analysis and analysis of DNA regulatory signals it was possible to distinguish metal-dependent members of COG0789, assign specificity towards particular metals to uncharacterized loci, and find new genes involved in the metal resistance, in particular, multicopper oxidase and copper chaperones, candidate cytochromes from the copper regulon, new cadmium transporters and, possibly, glutathione-S-transferases.ConclusionOur data indicate that the specificity of the COG0789 systems can be determined combining phylogenetic analysis and identification of DNA regulatory sites. Taking into account signal structure, we can adequately identify genes that are activated using the DNA bending-unbending mechanism. In the case of regulon members that do not reside in single loci, analysis of potential regulatory sites could be crucial for the correct annotation and prediction of the specificity.

NmlR ofNeisseria gonorrhoeae: a novel redox responsive transcription factor from the MerR family

A MerR-like regulator (NmlR -Neisseria merR-like Regulator) identified in the Neisseria gonorrhoeae genome lacks the conserved cysteines known to bind metal ions in characterized proteins of this family. Phylogenetic analysis indicates that NmlR defines a subfamily of MerR-like transcription factors with a distinctive pattern of conserved cysteines within their primary structure. NmlR regulates itself and three other genes in N. gonorrhoeae encoding a glutathione-dependent dehydrogenase (AdhC), a CPx-type ATPase (CopA) and a thioredoxin reductase (TrxB). An nmlR mutant lacked the ability to survive oxidative stress induced by diamide and cumene hydroperoxide. It also had > 50-fold lower NADH-S-nitrosoglutathione oxidoreductase activity consistent with a role for AdhC in protection against nitric oxide stress. The upstream sequences of the NmlR regulated genes contained typical MerR-like operator/promoter arrangements consisting of a dyad symmetry located between the -35 and -10 elements of the target genes. The NmlR target operator/promoters were cloned into a beta-galactosidase reporter system and promoter activity was repressed by the introduction of NmlR in trans. Promoter activity was activated by NmlR in the presence of diamide. Under metal depleted conditions NmlR did not repress P(AdhC) (or P(CopA)) promoter activity, but this was reversed in the presence of Zn(II), indicating repression was Zn(II)-dependent. Analysis of mutated promoters lacking the dyad symmetry revealed constitutive promoter activity which was independent of NmlR. Gel shift assays further confirmed that NmlR bound to the target promoters possessing the dyad symmetry. Site-directed mutagenesis of the four NmlR cysteine residues revealed that they were essential for activation of gene expression by NmlR.

Systematic targeted mutagenesis of Brucella melitensis 16M reveals a major role for GntR regulators in the control of virulence.

In order to identify transcriptional regulators involved in virulence gene control in Brucella melitensis, we generated a collection of 88 mutants in the AraC, ArsR, Crp, DeoR, GntR, IclR, LysR, MerR, RpiR, and TetR families of regulators. This collection was named LiMuR (library of mutants for regulators). We developed a method to test several mutants simultaneously in one animal in order to identify those unable to survive. This method, called the plasmid-tagged mutagenesis method, was used to test the residual virulence of mutants after 1 week in a mouse model of infection. Ten attenuated mutants, of which six and three belong to the GntR and LysR families, respectively, were identified and individually confirmed to replicate at lower rates in mice. Among these 10 mutants, only gntR10 and arsR6 are attenuated in cellular models. The LiMuR also allows simple screenings to identify regulators of a particular gene or operon. As a first example, we analyzed the expression of the virB operon in the LiMuR mutants. We carried out Western blottings of whole-cell extracts to analyze the production of VirB proteins using polyclonal antisera against VirB proteins. Four mutants produced small amounts of VirB proteins, and one mutant overexpressed VirB proteins compared to the wild-type strain. In these five mutants, reporter analysis using the virB promoter fused to lacZ showed that three mutants control virB at the transcriptional level. The LiMuR is a resource that will provide straightforward identification of regulators involved in the control of genes of interest.

Genetic control for light-induced carotenoid production in non-phototrophic bacteria

Carotenoids are naturally occurring yellow or orange pigments that serve as a protectant against photo-oxidative damages. Among the wide variety of producers, the prokaryotes generate a broad spectrum of carotenoids with diverse chemical structures that are expected to have a high potential in biotechnological applications. Bacterial carotenogenesis occurs in a constitutive or light-induced manner, which suggests the diversity of the regulatory mechanism. The mechanism for light-induced carotenoid production in non-phototrophic bacteria has been studied in detail in Myxococcus xanthus, a Gram-negative gliding bacterium. The complicated mechanism involves the activation of an extracytoplasmic function (ECF) sigma factor (CarQ), which leads to the sequestration of a MerR family transcriptional regulator (CarA) that represses the expression of the carotenoid biosynthesis genes in the dark. Recently, we identified another regulatory mechanism for light-induced carotenogenesis in Streptomyces coelicolor A3(2), a Gram-positive soil bacterium. In this organism, the transcription of the carotenoid biosynthesis gene cluster is specified by LitS, a photo-inducible ECF sigma factor. The evidence indicates that the photo-dependent transcription of litS is mediated by LitR, a MerR family transcriptional regulator. In addition, it is suggested that the conformational alteration of LitR upon receiving the illumination signal determines its binding to DNA. The carboxy-terminal domain of LitR contains a possible binding site for Vitamin B12, which may serve as a capturing apparatus for the illumination signal.

A Design for Life: Prokaryotic Metal-binding MerR Family Regulators

The MerR family of metal-binding, metal-responsive proteins is unique in that they activate transcription from unusual promoters and coordinate metals through cysteine (and in the case of ZntR, histidine) residues. They have conserved primary structures yet can effectively discriminate metals in vivo.

ThebldCDevelopmental Locus ofStreptomyces coelicolorEncodes a Member of a Family of Small DNA-Binding Proteins Related to the DNA-Binding Domains of the MerR Family

The bldC locus, required for formation of aerial hyphae in Streptomyces coelicolor, was localized by map-based cloning to the overlap between cosmids D17 and D25 of a minimal ordered library. Subcloning and sequencing showed that bldC encodes a member of a previously unrecognized family of small (58- to 78-residue) DNA-binding proteins, related to the DNA-binding domains of the MerR family of transcriptional activators. BldC family members are found in a wide range of gram-positive and gram-negative bacteria. Constructed DeltabldC mutants were defective in differentiation and antibiotic production. They failed to form an aerial mycelium on minimal medium and showed severe delays in aerial mycelium formation on rich medium. In addition, they failed to produce the polyketide antibiotic actinorhodin, and bldC was shown to be required for normal and sustained transcription of the pathway-specific activator gene actII-orf4. Although DeltabldC mutants produced the tripyrrole antibiotic undecylprodigiosin, transcripts of the pathway-specific activator gene (redD) were reduced to almost undetectable levels after 48 h in the bldC mutant, in contrast to the bldC+ parent strain in which redD transcription continued during aerial mycelium formation and sporulation. This suggests that bldC may be required for maintenance of redD transcription during differentiation. bldC is expressed from a single promoter. S1 nuclease protection assays and immunoblotting showed that bldC is constitutively expressed and that transcription of bldC does not depend on any of the other known bld genes. The bldC18 mutation that originally defined the locus causes a Y49C substitution that results in instability of the protein.

Global transcriptome analysis of the heat shock response of Shewanella oneidensis.

Shewanella oneidensis is an important model organism for bioremediation studies because of its diverse respiratory capabilities. However, the genetic basis and regulatory mechanisms underlying the ability of S. oneidensis to survive and adapt to various environmentally relevant stresses is poorly understood. To define this organism's molecular response to elevated growth temperatures, temporal gene expression profiles were examined in cells subjected to heat stress by using whole-genome DNA microarrays for S. oneidensis. Approximately 15% (n = 711) of the total predicted S. oneidensis genes (n = 4,648) represented on the microarray were significantly up- or downregulated (P < 0.05) over a 25-min period after shift to the heat shock temperature. As expected, the majority of the genes that showed homology to known chaperones and heat shock proteins in other organisms were highly induced. In addition, a number of predicted genes, including those encoding enzymes in glycolysis and the pentose cycle, serine proteases, transcriptional regulators (MerR, LysR, and TetR families), histidine kinases, and hypothetical proteins were induced. Genes encoding membrane proteins were differentially expressed, suggesting that cells possibly alter their membrane composition or structure in response to variations in growth temperature. A substantial number of the genes encoding ribosomal proteins displayed downregulated coexpression patterns in response to heat stress, as did genes encoding prophage and flagellar proteins. Finally, a putative regulatory site with high conservation to the Escherichia coli sigma32-binding consensus sequence was identified upstream of a number of heat-inducible genes.

Functional Analysis of SoxR Residues Affecting Transduction of Oxidative Stress Signals into Gene Expression

SoxR protein, a member of the MerR family of transcriptional activators, mediates a global oxidative stress response in Escherichia coli. Upon oxidation or nitrosylation of its [2Fe-2S] centers SoxR activates its target gene, soxS, by mediating a structural transition in the promoter DNA that stimulates initiation by RNA polymerase. We explored the molecular basis of this signal transduction by analyzing mutant SoxR proteins defective in responding to oxidative stress signals in vivo.We have confirmed that the DNA binding domain of SoxR is highly conserved compared with other MerR family proteins and functions in a similar manner to activate transcription. Several mutations in the dimerization domain of SoxR disrupted intersubunit communication, and the resulting proteins were unable to propagate redox signals to the soxS promoter. Mutations scattered throughout the polypeptide yielded proteins that were under-responsive to in vivo redox signals, which indicates that the redox properties of the [2Fe-2S] centers are influenced by global protein structure. These findings indicate that SoxR functions as a redox-responsive molecular switch in which subunit interactions transduce a subtle alteration in oxidation state into a profound change in DNA structure.

The Structural Mechanism for Transcription Activation by MerR Family Member Multidrug Transporter Activation, N Terminus

Transcription regulators of the MerR family respond to myriad stress signals to activate sigma70/sigmaA-targeted genes, which contain suboptimal 19-bp spacers between their -35 and -10 promoter elements. The crystal structure of a BmrR-TPP(+)-DNA complex provided initial insight into the transcription activation mechanism of the MerR family, which involves base pair distortion, DNA undertwisting and shortening of the spacer, and realignment of the -35 and -10 boxes. Here, we describe the crystal structure of MerR family member MtaN bound to the mta promoter. Although the global DNA binding modes of MtaN and BmrR differ somewhat, homologous protein-DNA interactions are maintained. Moreover, despite their different sequences, the mta promoter conformation is essentially identical to that of the BmrR-TPP(+)-bound bmr promoter, indicating that this DNA distortion mechanism is common to the entire MerR family. Interestingly, DNA binding experiments reveal that the identity of the two central bases of the mta and bmr promoters, which are conserved as either a thymidine or an adenine in nearly all MerR promoters, is not important for DNA affinity. Comparison of the free and DNA-bound MtaN structures reveals that a conformational hinge, centered at residues N-terminal to the ubiquitous coiled coil, is key for mta promoter binding. Analysis of the structures of BmrR, CueR, and ZntR indicates that this hinge may be common to all MerR family members.

A General Strategy to Convert the MerR Family Proteins into Highly Sensitive and Selective Fluorescent Biosensors for Metal Ions

The MerR family metal-regulatory proteins are converted into fluorescent biosensors that can detect different metal ions with high sensitivity and selectivity.

An Atypical Linear Cu(I)−S2 Center Constitutes the High-Affinity Metal-Sensing Site in the CueR Metalloregulatory Protein

CueR is a copper-responsive genetic switch that regulates transcription of genes encoding the primary copper-exporting system in E. coli, CopA and CueO. Although a member of the MerR family of regulatory proteins, CueR has four cysteines in an array that is distinct from those in Hg(II)-sensing MerR or in Zn-sensing ZntR. A recent crystal structure showed one copper atom in CueR bound to cysteines Cys112 and Cys120 in a linear CuS2 structure, but left open the questions of whether the other half of the CueR dimer has the same structure, and of whether these structures depend on the two additional C-terminal cysteines. Metal binding, transcription runoff, and cysteine modification studies show that only Cys112 and Cys 120 are necessary and sufficient to make the transcriptionally active Cu-sensing site. X-ray absorption spectroscopy shows that this site binds Cu(I) in a strictly linear CuS2 site in solution, a structure that is rarely observed in copper proteins. This structure does not depend either on additional metal loading or upon the presence of additional C-terminal cysteine ligands and is well suited for an ultrasensitve receptor site that discriminates against the binding of other metal ions.